1. Field of the Invention
This invention is directed to an apparatus and method for the measurement of photoluminescent lifetimes. More specifically, the invention concerns a low cost portable frequency-domain phase fluorometer which operates at a frequency of up to 200 MHz without the need for a cross-correlation detection.
2. Prior Art
The contents of all cited references including literature references, issued patents, published patent applications as cited throughout this application are readily available to those skilled in the art and are hereby expressly incorporated herein by reference as though they were set forth herein in their entirety.
Anghel, F., C. Iliescu, K. T. V. Grattan, A. W. Palmer and Z. Y. Zhang, xe2x80x9cFluorescent-Based Lifetime Measurement Thermometer for Use at Subroom Temperatures (200-300 K)xe2x80x9d, Rev. Sci. Instrum. 66, 2611-2614, 1995.
Bambot, S., R. Holavanahali, J. R. Lakowicz, G. M. Carter and G. Rao, xe2x80x9cPhase Fluorometric Sterilizable Optical Oxygen Sensorxe2x80x9d, Biotechnology and Bioengineering 43, 1139-1145, 1994.
Chang, Q., L. Randers-Eichhorn, J. R. Lakowicz, G. Rao, xe2x80x9cSteam-Sterilizable Fluorescence Lifetime-Based Sensing Film for Dissolved Carbon Dioxidexe2x80x9d, Biotechnol. Prog. 14, 326-331, 1998.
Gratton, E. and M. Limkeman, xe2x80x9cA Continuously Variable Frequency Cross-Correlation Phase Fluorometer with Picosecond Resolution,xe2x80x9d Biophys. J., 315-324, 1983.
Gruber, W. R., P. O""Leary, O. S. Wolfbeis, xe2x80x9cDetection of Fluorescence Lifetime Based on Solid-State Technology, and Its Application to Optical Sensingxe2x80x9d, Proc. SPIE 2388, 148-158, 1995.
Gryczynski, I., J. Kusba, J. R. Lakowicz, xe2x80x9cEffect of Light Quenching on the Emission Spectra and Intensity Decays of Fluorophore Mixturesxe2x80x9d, J. Fluorescence 7, 167-183, 1997.
Holavanahali, R., M. Romauld, G. M. Carter, G. Rao, J. Sipior, J. R. Lakowicz and J. D. Bierlein, xe2x80x9cDirectly Modulated Diode Laser Frequency-Doubled in a KTP Waveguide as an Excitation Source for CO2 and O2 Phase Fluorometric Sensorsxe2x80x9d, J. Biomed. Optics 1, 124-130, 1996.
Hoist, G. A., T. Koster, E. Voges, D. Lubbers, xe2x80x9cFLOX an Oxygen-Flux-Measuring System Using a Phase-Modulation Method to Evaluate the Oxygen-Dependent Fluorescence Lifetimexe2x80x9d, Sens. Actuators B 29, 231-9, 1995.
Lakowicz, J. R., Principles of Fluorescence Spectroscopy. Plenum Press, New York, 1983.
Lakowicz, J. R. and I. Gryczynski, xe2x80x9cFrequency-Domain Fluorescence Spectroscopyxe2x80x9d, in Topics in Fluorescence Spectroscopy, Vol. 1 Techniques, (J. R. Lakowicz, Ed.), 293-335, 1991.
Lakowicz, J. R., G. Laczko, I. Gryczynski, xe2x80x9c2-GHz Frequency-Domain Fluorometerxe2x80x9d, Rev. Sci. Instrum. 57, 2499-2506, 1986.
Lakowicz, J. R. and B. Maliwal, xe2x80x9cConstruction and Performance of a Variable-Frequency Phase-Modulation Fluorometerxe2x80x9d, Biophysical Chemistry 21, 61-78, 1985.
Lakowicz, J. R. and B. Maliwal, xe2x80x9cOptical Sensing of Glucose Using Phase-Modulation Fluorimetryxe2x80x9d, Anal. Chim. Acta. 271, 155-164, 1993.
Levy, R., E. F. Guignon, S. Cobane, E. St. Louis and S. M. Femandez, xe2x80x9cCompact, Rugged and Inexpensive Frequency-Domain Fluorometerxe2x80x9d, SPIE 2980, 81-89, 1997.
Murtagh, M. T ., D. E. Acklev, M. R. Shahriari, xe2x80x9cDevelopment of a Highly Sensitive Fiber Optic O2/DO Sensor Based on a Phase Modulation Techniquexe2x80x9d, Electronics Letters 32, 477-479, 1996.
Ozinskas, A, H. Malak, J. Joshi, H. Szmacinski, J. Britz, R. Thompson, P. Koen and J. R. Lakowicz, xe2x80x9cHomogeneous Model Immunoassay of Thyroxine by Phase-Modulation Fluorescence Spectroscopyxe2x80x9d, Anal. Biochem. 213, 264-270, 1993.
Spencer, R. D. and G. Weber, xe2x80x9cMeasurement of Sub-Nanosecond Fluorescence Lifetime with a Cross-Correlation Phase Fluorometerxe2x80x9d, Ann. N. Y. Acad. Sci. 158, 361-376, 1969.
Sipior, J., G. Carter, J. R. Lakowicz, G. Rao, xe2x80x9cSingle Quantum Well Light Emitting Diodes Demonstrated as Excitation Sources for Nanosecond Phase-Modulation Fluorescence Lifetime Measurementsxe2x80x9d, Rev. Sci. Instrum. 67, 3795-3798, 1996.
Szmacisnki, H., J. R. Lakowicz, xe2x80x9cOptical Measurements of pH Using Fluorescence Lifetimes and Phase-Modulation Fluorometryxe2x80x9d, Anal. Chem. 65, 1668-1674, 1993.
Thompson, R. B., Z. Ge, M. W. Patchan and C. A. Fierke, xe2x80x9cPerformance Enhancement of Fluorescence Energy Transfer-Based Biosensors by Site-Directed Mutagenesis of the Transducerxe2x80x9d, J. Biomed. Optics 1, 131-137,1996.
Zhang, Z., K. T. V. Grattan, A. W. Palmer, xe2x80x9cA Novel Signal Processing Scheme for a Fluorescence Based Fiber-Optic Temperature Sensorxe2x80x9d, Rev. Sci. Instrum. 62, 1735-42, 1991.
U.S. Patents
Numerous chemical and biochemical research tools, remote sensing devices, and immunodiagnostic test methods are based on some form of photoluminometric analysis (e.g., fluorometry and phosphorimetry). Although the disclosure herein primarily focuses on a fluorometric apparatus and method of analysis, the terms used herein such as fluroescence, fluorophore and fluorometer, are to be construed to include the meaning of phosphorescence, phosphor and phosphorimeter, respectively.
Fluorometry offers a wide range of advantages over other spectroscopic methods (e.g., colorimetry). These advantages include low detection limits and the potential for minimally invasive measurements in biological samples. However, simple intensity based methods are prone to artifacts because any change in fluorescence intensity, regardless of origin, can flaw the analysis. Specifically, intensity measurements can suffer from interferences caused by light scattering, variations in the intensity of the source or excitation light, photobleaching, contaminating chromophores, or changes in the collection geometries. To circumvent the limitations of intensity measurements, Lakowicz and other researchers developed methods based on measuring the fluorescence lifetime of a fluorophore in the time or frequency domain (Lakowicz et al. (1986); Zhang et al. (1991); Gruber et al. (1995); Holst et al. (1995); and Murtagh et al. (1996)). Fluorescence lifetime analysis has been used, for example, to study: rotational and molecular diffusion; energy transfer kinetics and other excited state reactions; and collisional quenching (Lakowicz, 1983). Fluorescence lifetime analysis has also been used in the development of immunoassays (Ozinskas, 1993); sensors for the measurement of pH (Szmacisnki et al., 1993); temperature (Anghel et al., 1995); glucose (Lakowicz and Maliwal, 1993); metal ions (Thompson et al., 1996); oxygen (Bambot et al., 1994); and carbon dioxide (Holavanahali et al., 1993).
Various methods for measuring fluorescence lifetimes are common to the art and include both time domain and frequency domain methods. In time domain methods, the fluorescence lifetime of a sample is determined from an analysis of the fluorescence decay that is elicited by a pulsed excitation. In general, a sample is excited with a brief pulse of light and the time-dependent decay in fluorescence intensity is measured. However, the measurement of the decay in fluorescence intensity is difficult as light sources typically yield pulses with durations of several nanoseconds. As a result, one must either correct for the pulse width or select an alternative light source which can yield pulses of a duration shorter than the average lifetime being measured. Generally such pulsed picosecond light sources are not only expensive but add to the technical complexity of the system. A further difficulty of time domain methods is the need to measure the entire duration of the time-resolved fluorescence decay. This difficulty is generally minimized by exciting the sample with repetitive pulses that are spaced at time intervals greater than a factor of five times the decay time, to avoid overlap of the decay pulses. However, if repetitive pulses are used, the decay in fluorescence intensity must be reconstructed using either a stroboscopic or photon counting method (Lakowicz, 1983). Further, one must also correct for the finite width of the light pulse and the response time of the detection system when using a photon counting technique (e.g., Time-Correlated Single Photon Counting Technique (TCSPC)) in order to obtain the true fluorescence decay pulse.
Frequency domain or phase-modulation methods do not require corrections for the finite width of the excitation pulses or the time response of the detection system. Further, frequency domain or phase-modulation methods have the advantage of lower cost electronics than that required for decay time measurement as the excitation light pulse can be longer in duration. Typically, in frequency domain or phase modulation methods, the intensity of the excitation light is pulsed or modulated sinusoidally, resulting in fluorescence at the same circular frequency as the excitation light. However, due to the finite duration of the fluorophore""s excited state, the emitted fluorescence lags in phase by an angle, xcfx86, as compared to the circular frequency of the excitation light. In addition, the depth of modulation compared to the excitation is also reduced. A demodulation factor, m, is defined by the equation:
m=(B/A)/(b/a)xe2x80x83xe2x80x83(1);
where xe2x80x9caxe2x80x9d is the average value of the emitted fluorescence; xe2x80x9caxe2x80x9d is the average value of the excitation light; xe2x80x9cBxe2x80x9d is the amplitude of the peak emission above its average value; and xe2x80x9cbxe2x80x9d is the amplitude of the peak excitation above its average value.
The circular frequency of the excitation light xcfx89 can be expressed by the equation:
xcfx89=(2xcfx80) (frequency)xe2x80x83xe2x80x83(2);
where the frequency is expressed in Hz. Typically, both the phase angle xcfx86 and the demodulation factor m which corresponds to the reduction in the depth of modulation compared to the excitation are measured and used to calculate the phase xcfx84p and modulation xcfx84m lifetimes of the sample, respectively. This provides two independent measurements of the fluorescence lifetime, giving an added degree of robustness to the measurement. The phase xcfx84p lifetime can be calculated using the equation:
xcfx84p=(xcfx89xe2x88x921)(tan xcfx86)xe2x80x83xe2x80x83(3);
whereas, the modulation xcfx84m lifetime can be calculated using the equation:
xcfx84m=(xcfx89xe2x88x921)[(1/m2)xe2x88x921]xc2xdxe2x80x83xe2x80x83(4).
Multifrequency measurements in combination with non-linear least squares curve fitting techniques are used in multi-exponential decay analyses of heterogeneous samples. Plots of phase angle vs. modulation frequency, or demodulation vs. modulation frequency are used in fluorescence lifetime analysis. This multi-exponential decay analysis is based on known theoretical considerations by and between phase angle and modulation with fluorescence lifetime (Lakowicz and Gryczynski, 1991).
Frequency domain or phase-modulation methods have also been successfully used in remote sensing applications. A variety of sensors have been developed which are responsive to changes in environmental factors such as temperature and pH. Although the time dependent emission of fluorescence by a fluorophore can usually be characterized by a single exponential decay constant, multiple decay constants can result from either changes in a fluorophore""s environment, or as the result of various excited state processes. As with heterogeneous fluorescent samples, multifrequency measurements in combination with non-linear least squares curve fitting techniques can be used in the analysis. Alternatively, an average lifetime measurement can provide a suitable metric that correlates with the environmental factor of interest (Levy et al., 1997). Further, fluorescence lifetime based sensors avoid the problems associated with photobleaching and excitation source intensity which are common to intensity based sensor designs (Lakowicz, 1984).
To date the major drawback of fluorescence lifetime analysis is the component cost of the system. Even the lower component cost of phase-modulation as compared to pulse systems is beyond the budget of most labs, especially for the measurement of nanosecond lifetimes. Less expensive and simpler instrument designs can be used with long lifetime fluorophores but these fluorophores do not have the sensing capability that conventional nanosecond probes offer. Fluorophores with lifetimes on the order of hundreds of nanoseconds to a few microseconds have been measured with low-cost lock-in amplifier based systems, albeit only for phase angle measurements (Bambot et al. (1994)).
The components and costs associated with the construction of a variable frequency phase-modulation fluorometer are well known. For example see Lakowicz and Maliwal (1985). The instrument described provides modulation frequencies from 1 to 200 MHz and measures phase angles and demodulation factors using a cross-correlation detection method. The cross-correlation technique is well known to those skilled in the art and is described in Spencer and Weber, xe2x80x9cMeasurement of Sub-Nanosecond Fluorescence Lifetime with a Cross-Correlation Phase Fluorometerxe2x80x9d (1969). The basic cross-correlation phase fluorometer includes a light source (helium-cadmium laser), an electro-optic modulator, two frequency synthesizers, two radio frequency power amplifiers, and various optical and electronic parts for a component cost in excess of US $50,000. Current commercially available multifrequency fluorometers are priced on the order of US $100,000.
A diode-laser based cross-correlation phase fluorometer suitable for single frequency sensor applications has recently been described in the article by Levy et al. (1997). This system is an inexpensive alternative to the multifrequency fluorometers described above. In contrast to conventional cross-correlation methods in which the signal frequency is generated as a difference in the frequency of two high frequency sources, the instrument described in Levy et al. (1997) uses a single sideband technique in which the signal frequency is generated as the sum of two different frequencies. However, this approach is limited with respect to multifrequency applications. Specifically, given their method of down-conversion, corrections for optically or electronically induced phase shifts common to all phase fluorometers have to be made at each tested frequency. Furthermore, the instrument described in Levy et al. (1997) provides a lifetime measurement based on just phase angle.
The high cost and complexity of performing lifetime measurements at multiple frequencies presented an opportunity for the development of an inexpensive and practical instrument for the measurement of fluorescence lifetimes using a phase-modulation method. Recent advances in digital signal processors has produced a new class of lock-in amplifiers and provided the core component to the present invention. The SR844 RF lock-in amplifier (Stanford Research Systems, Sunnyvale, Calif.) is commercially available and representative of this new class. While most lock-in amplifiers have a maximum frequency of 100 kHz, the SR844 can measure up to 200 MHz.
The present invention relates to an apparatus and method for measuring nanosecond photoluminescence lifetimes. The basic apparatus presents a low cost alternative for the measurement of photoluminescence lifetimes using a multiple frequency phase sensitive detection technique. Unlike the prior art, the use of multiple mixers in a lock-in amplifier to get baseband signals in quadrature obviates the need to re-calibrate at each frequency, providing a much simpler method for multifrequency applications. Further, lifetime analysis with the present invention are based on two independent measurements, phase angle and modulation. It is the development of high frequency lock-in amplifiers coupled with the emergence of a wide range of low cost light sources (e.g., LEDs) which can be modulated at high frequencies, that have enabled the design and construction of a variable frequency phase-modulation fluorometer in accordance with the present invention at a component cost of under US $10,000.
As used herein, photoluminescence means any of a group of processes whereby a material is excited by radiation such as light, raised to an excited electronic or vibronic state, and subsequently re-emits that excitation energy as a photon of light. These processes include fluorescence and phosphorescence. Fluorescent emissions accompany the descent of excited state electrons from paired xe2x80x9csingletxe2x80x9d states to lower states of the same multiplicity; an xe2x80x9callowedxe2x80x9d quantum-mechanics transition. In contrast, phosphorescent emissions accompany the descent of excited state electrons from unpaired xe2x80x9ctripletxe2x80x9d states to lower states of a different multiplicity; a xe2x80x9cforbiddenxe2x80x9d quantum-mechanics transition.
Since in any given sample more than one lifetime can be present, the term xe2x80x9clifetimexe2x80x9d means and includes the term xe2x80x9clifetimesxe2x80x9d and the term xe2x80x9clifetimesxe2x80x9d means and includes the term xe2x80x9clifetimexe2x80x9d, so as to include all possibilities under the circumstances, including multiple lifetimes that are present in the particular sample under consideration.
The present invention provides a low cost alternative to the complex multifrequency phase-modulation instruments that are commercially available. The level of complexity and component cost of an instrument according to the present invention place it within the economic reach of most laboratories. Accordingly, the ability to measure fluorescence lifetimes using a multiple frequency phase-modulation technique no longer requires expensive apparatus employing complex and expensive methods. The basic apparatus according to a specific embodiment of the present invention comprises an optical bench, a lock-in amplifier, two bias tees, a blue light emitting diode (LED), a reference standard, a filter and a photomultiplier tube and has a component cost of less than US $10,000. Additionally, sample holders such as cuvettes, reaction chambers and flow cells can also be used. Similarly, photodiodes can be used in lieu of the photomultiplier tube.
The method of the present invention comprises the steps of (i) providing an AC reference signal; (ii) providing a DC bias signal; (iii) biasing the AC reference signal with the DC bias signal to produce a biased AC input signal; (iv) modulating a light source at a frequency of said biased AC input signal to produce modulated exciting light having a wavelength capable of exciting a photoluminescent species; (v) disposing a photoluminescent species in a sample holder; (vi) detecting modulated light emission from the photoluminescent species and producing a modulated electrical emission signal; (vii) processing the modulated electrical emission signal to obtain values indicative of the photoluminescent lifetime of the photoluminescent species.
It is another object of the present invention to provide a phase-modulation fluorometer which lends itself to portability and field use. It is a further object of the present invention to provide for use with fiber optics, making the phase-modulation fluorometer ideal for use with fluorescence lifetime based sensors. It is a further object of the present invention that it is portable, consumes little power, and is easily configured for use with fiber optics, making it ideal for field use with fluorescent lifetime based sensors.
In a preferred specific embodiment of the present invention employing heterodyning, a lock-in amplifier provides an AC reference signal and a DC bias signal. A bias tee is used to bias the AC reference signal against the DC bias signal producing a biased AC input signal. An output of the bias tee or the biased AC input signal is used to modulate a blue light emitting diode (LED) A modulated radiant output of the LED is used to excite a photoluminescent species that has been disposed in a sample holder. A modulated light emission from the photoluminescent species in the sample holder is detected by a photomultiplier tube producing a modulated electrical emission signal. A second bias tee is used to split the modulated electrical emission signal into its AC and DC component signals. The DC component is measured, and the AC component output is mixed with an AC reference signal whereby the phase and amplitude information are shifted to a lower frequency signal. The above lock-in amplifier is also used to sample, filter, apply offsets and analyze the lower frequency signal to obtain a DC value wherein the DC value is indicative of the value(s) of the photoluminescent lifetime(s) of the photoluminescent species that was disposed in the sample holder. No external signal processing or heterodyning is required of this lock-in amplifier based system.
In an alternative specific embodiment employing homodyning, after splitting the modulated electrical emission signal into its AC and DC component signals and measuring the DC component, the AC component is mixed with AC reference signals and the resulting signal is filtered and analyzed whereby the phase and amplitude information are expressed as two DC values that are indicative of the value(s) of the fluorescence lifetime(s) of a photoluminescent species.
In a further alternative specific embodiment employing direct sampling, after splitting the modulated electrical emission signal into its AC and DC component signals and measuring the DC component, the AC component is filtered, sampled and analyzed whereby the phase and amplitude information are expressed as two DC values that are indicative of the value(s) of the fluorescence lifetime(s) of a photoluminescent species.
It is expressly noted herein that the values obtained directly by the apparatus and methods of the present invention are not the lifetime(s) themselves and that these values require further computer analysis to interpret these values to thereby obtain the actual lifetime(s).
In yet another specific embodiment of the present invention involving heterodyning, the system further comprises a bifurcated fiber optic bundle having two proximal arms and a common end optically coupled to a porous fiber, solid or gel matrix (hereinafter a xe2x80x9csensor patchxe2x80x9d) containing one or more photoluminescent species. In this embodiment, a distal end of one of the proximal arms is optically coupled to a modulated light source; a distal end of the other proximal arm is optically coupled to a detector; and a common end of the bifurcated fiber optic containing the sensor patch is either disposed in a sample holder or optically coupled to the sample holder. The method of this alternative specific embodiment comprises the steps of (i) generating an AC reference signal; (ii) generating a DC bias signal; (iii) biasing the AC reference signal with the DC bias signal to produce a biased AC input signal; (iv) modulating a light source at a frequency of said biased AC input signal to produce modulated exciting light having a wavelength capable of exciting a photoluminescent species; (v) optically coupling a distal end of a first proximal arm of a bifurcated fiber optic to the modulated light source; (vi) disposing a common end of the bifurcated fiber optic which has at least one photoluminescent species disposed in a sensor patch on its end into a sample holder or optically coupling the common end of the bifurcated fiber optic to the sample holder; (vii) disposing an analyte or sample of interest into the sample holder or introducing a flow carrying the analyte or sample of interest into the sample holder; (viii) exciting the photoluminescent species with the modulated light source light; (ix) transmitting the modulated source light through the bifurcated fiber optic from the distal end of the proximal arm optically coupled to the modulated light source to the common end of the fiber optic to produce a corresponding emission from the photoluminescent species disposed on the common end of the fiber optic; (x) transmitting the modulated emission from the photoluminescent species disposed on the common end of the fiber optic to the distal end of a second proximal arm of the fiber optic; (xi) optically coupling a distal end of the remaining proximal arm of the bifurcated fiber optic to a detector; (xii) detecting the emission from the distal end of the remaining proximal arm of the bifurcated fiber optic, producing a modulated emission signal; (xiii) splitting the modulated emission signal into its AC and DC components, measuring the DC component, and mixing the AC component with an AC reference signal whereby the phase and amplitude information are shifted to a lower frequency; and (xiv) sampling, filtering, applying offsets and analyzing said lower frequency signal to obtain a DC value wherein the DC value is indicative of a value of the photoluminescent lifetime of the photoluminescent species.
The use of the bifurcated optic bundle in the alternative specific embodiment obviates the need for the optical bench such as described in the first preferred specific embodiment involving heterodyning. Further, the bifurcated optic bundle optically couples both the modulated light source and the detector with a photoluminescent species disposed at the common end of the optic bundle. Signal processing is performed as described above.
In yet a further alternative specific embodiment again using the bifurcated fiber optic and further employing homodyning, and as previously described, after splitting the modulated electrical emission signal into its AC and DC component signals and measuring the DC component, the AC component is mixed with AC reference signals and the resulting signal is filtered and analyzed whereby the phase and amplitude information are expressed as two DC values that are indicative of the value(s) of the fluorescence lifetime(s) of a photoluminescent species.
In yet a further alternative specific embodiment again using the bifurcated fiber optic and further employing direct sampling, and as previously described, after splitting the modulated electrical emission signal into its AC and DC component signals and measuring the DC component, the AC component is filtered, sampled and analyzed whereby the phase and amplitude information are expressed as two DC values that are indicative of the value(s) of the fluorescence lifetime(s) of a photoluminescent species.
In another specific alternative embodiment using the bifurcated fiber optic, the photoluminescent species is immobilized on the common end of the fiber optic.
In a further specific alternative embodiment using the bifurcated fiber optic, the sensor patch is eliminated and one or more photoluminescent species are disposed in the sample holder with an analyte or sample of interest, or a flow carrying the photoluminescent species and the analyte or sample of interest is introduced into the sample holder.
In a further specific embodiment, the entire lock-in amplifier based system can be controlled by any standard personal computer through a GPIB, USB, serial or similar connection on the lock-in amplifier.
In a further specific embodiment the present invention is battery operated and portable making it ideal for use with photoluminescent lifetime based sensors.